The present invention relates to methods for the detection of the presence or absence of nucleic acid sequences which are characteristic of pathogens and the like as well as of gene variations and mutations including those relating to the human leukocyte antigen (HLA) which is of interest in the field of human transplantation.
The HLA locus is highly polymorphic in nature. As disclosed in the Nomenclature for Factors of the HLA System 1996 (Tissue Antigens 1997: 49:297-321), there are 83 HLA-A alleles, 186 HLA-B alleles, 42 HLA-C alleles, 184 HLA-DRB1 alleles, 11 DRB3 alleles, 8 DRB4 alleles, 12 DRB5 alleles, 18 DQA1 alleles and 31 DQB1 alleles, with new alleles being discovered continuously. All HLA-A, -B, and -C alleles have similar sequences. The same holds for DRB1, DRB3, DRB4 and DRB5 sequences. Because of these similarities, very often when a primer pair is used in the practice of polymerase chain reaction sequence-specific priming (PCR-SSP), two or more alleles will be amplified. Therefore, for each allele to have a unique PCR-SSP pattern many pairs of primers must be used. Accordingly, in clinical use of PCR-SSP for HLA typing there exists a desire to use a limited number of PCR reactions to achieve as much resolution as possible whereby the number of alleles amplified by a pair of primers would be reduced (i.e., the specificity of the primers increased). Simultaneously, all of the primer pairs must have optimal annealing temperatures within a very restricted range.
PCR requires a pair of primers flanking the region on the DNA template for that region to be amplified. The ability of a primer to anneal to the desired sequence depends on the length of the primer and the annealing temperature set in the PCR thermocycling program. The longer the primer, the higher the annealing temperature it needs to achieve specific amplification of a DNA sequence. If the annealing temperature for a PCR is above the optimal range for the primer to anneal to its target, little or no amplification will occur. If the annealing temperature for a PCR is below the optimal range for the primer to anneal to its target, non-specific amplification will occur. PCR-SSP uses a balance between primer length and annealing temperature to achieve the specificity of the primer-directed sequence amplification. This technique can be used to characterize the sequence on the target DNA template--if amplification occurs, the template DNA contains the same sequences as the primers used; if no amplification occurs, the sequences on the template DNA are different from the primer sequences. Of interest to the present application are the disclosures of Olerup et al., Tissue Antigens 41: 119-134 (1993) and Bunce et al., Tissue Antigens 43: 7-17 (1994) which teach methods of PCR-SSP for HLA typing.
Newton et al., U.S. Pat. No. 5,595,890 disclose PCR diagnostic methods for typing including molecular typing of HLA using PCR-SSP. According to this method, an unknown allele is assigned based on the pattern of positive or negative reactions from multiple PCR. The methods disclosed by Newton are limited in their effectiveness for HLA typing, however, due to the high degree of polymorphism in HLA as described above. As a consequence two primers, each with specific sequences, frequently amplify many HLA alleles, thus increasing the required number of PCR in order to assign an unknown allele.
Accordingly, there exists a desire in the art for improved methods of PCR-SSP based molecular typing whereby the specificity of the typing can be increased so as to reduce the number of PCR reactions required for each typing.